Alzheimer's disease (AD) is clinically characterized by progressive memory loss and decline of cognitive functions and histopathologically by extracellular deposition of fragments (amyloid β (Aβ) peptides) of the amyloid precursor protein and intracellular deposits of hyperphosphorylated tau protein in neurofibrillary tangles. These fragments are generated by subsequent cleavages of two aspartic proteases BACE1 and presenilin 1, resulting in the liberation of Aβ peptides of various lengths (Aβ 1-38/40/42).
There is evidence that formation of, in particular, aggregated Aβ 1-42 contributes to synaptic dysfunction and oxidative stress that results in neuronal degeneration. One source of the oxidative stress is the formation of oxidative species arising from the conversion of nitric oxide to peroxynitrite.
The NOS2 gene encoding the inducible form of the nitric oxide synthase (iNOS) is one of three NOS proteins that generate NO in the brain. It has been shown that iNOS is upregulated in neurons and astrocytes in response to degenerative and inflammatory stimuli in Alzheimer's disease, potentially aggravating disease progression2,3. Despite potentially being involved in the aggravating of disease progression in AD, iNOS is a pro-inflammatory mediator that is upregulated not only in AD, but in many age-related diseases (see, e.g., Chung H Y, Cesari M, Anton S, Marzetti E, Giovannini S, Seo A Y, Carter C, Yu B P, Leeuwenburgh C. Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev. 2009 January; 8(1):18-30. Epub 2008 Jul. 18).
iNOS produces large amounts of NO for prolonged periods of time resulting in reactive nitrogen intermediates and thereby exerts effects on mitochondrial respiration4, enzyme activity5 neuronal cell death6-9 and induction of apoptosis10.
AD lesions reveal the pathological pattern of oxidative and nitrosative injury, especially the posttranslational modifications of cysteine and tyrosine residues11-15. One of the modifications is S-nitrosylation, or covalent reaction of NO with specific protein thiol groups, leading to protein misfolding and neurotoxicity16. In addition, conversion of protein tyrosine residues to 3′-nitrotyrosine have been found under pathological conditions and result in changes in enzyme activities, protein degradation and immune responses17.
Furthermore, it appears that an immunization using Aβ 1-42 resulted in a clearance of amyloid plaques in patients with AD, but this clearance did not prevent progressive neurodegeneration (Holmes C, Boche D, Wilkinson D, Yadegarfar G, Hopkins V, Bayer A, Jones R W, Bullock R, Love S, Neal J W, Zotova E, Nicoll J A. Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of a randomized, placebo-controlled phase I trial. Lancet. 2008 Jul. 19; 372(9634):216-23). Thus, it is under discussion in the state of the art whether an anti-amyloid therapy would improve the situation in patients.
Attempts have been undertaken to improve the situation in AD using anti-inflammatory agents, but the clinical trials failed.
WO 99/26657 and WO 98/09653 describe iNOS inhibitors and their prospective uses in, amongst others, AD. U.S. Pat. No. 7,300,955 describes the combined use of an inhibitor of formation or release of β-amyloid and a nitric oxide releaser for the treatment or prevention of Alzheimer's disease. U.S. Pat. No. 7,371,770 describes the use of an inhibitor of the formation of β-amyloid peptide in the treatment of AD.
In summary, researchers in Alzheimer's disease have identified five strategies as possible interventions against amyloid:    a) β-Secretase inhibitors. These work to block the first cleavage of APP outside of the cell.    b) γ-Secretase inhibitors (e.g. Semagacestat). These work to block the second cleavage of APP in the cell membrane and would then stop the subsequent formation of Aβ and its toxic fragments.    c) Selective Aβ42 lowering agents (e.g. Tarenflurbil). These modulate γ-secretase to reduce Aβ42 production in favor of other (shorter) Aβ versions.    d) Immunotherapies. These stimulate the host immune system to recognize and attack Aβ or provide antibodies that either prevent plaque deposition or enhance clearance of plaques.    e) Anti-aggregation agents based on the chemical structure of apomorphine. These molecules were found to interfere with Aβ1-40 fibrillization through oxidative processes (Lashuel H A, Hartley D M, Balakhaneh D, Aggarwal A, Teichberg S, Callaway D J E (2002). “New class of inhibitors of amyloid-beta fibril formation. Implications for the mechanism of pathogenesis in Alzheimer's disease”. J Biol Chem 277 (45): 42881-42890). These prevent Aβ fragments from aggregating or clear aggregates once they are formed (Michael H. Parker, Robert Chen, Kelly A. Conway, Daniel H. S. Lee; Chi Luoi, Robert E. Boyd, Samuel O. Nortey, Tina M. Ross, Malcolm K. Scott, Allen B. Reitz (2002). “Synthesis of (+)-5,8-Dihydroxy-3R-methyl-2R(dipropylamino)-1,2,3,4-tetrahydro-naphthalene: An Inhibitor of β-Amyloyid1-42 Aggregation”. Bioorg. Med. Chem. 10 (11): 3565-3569).
There is some indication that supplementation of the hormone melatonin may be effective against amyloid.
Despite the numerous attempts in the state of the art to identify effective targets to improve the situation with respect to the disease progression in AD, no satisfactory progresses have been made so far. It is therefore an object of the present invention, to provide such a new target that may serve as a promising approach for an improved treatment in AD. This target shall be used in assays to find new medicines for AD, and also to provide effective diagnostic assays for the disease. Further objects and advantages will become apparent to the person of skill when reading the following more detailed description of the present invention.
In a preferred first aspect of the present invention, the invention relates to a method for identifying an inhibitor of the aggregation of amyloid-β peptide (Aβ), comprising the steps of a) contacting at least one Aβ-peptide and/or the nitrated forms thereof with at least one candidate inhibitor that potentially specifically binds to a region in said Aβ-peptide capable of being nitrated, and b) detecting said inhibitor specifically binding to said region in said Aβ-peptide through detecting a lack of or a reduced aggregation of said at least one Aβ-peptide. Preferably, said Aβ-peptide is selected from 1-38, 1-40, and 1-42, and the nitrated forms thereof.
In a preferred second aspect of the present invention, the invention relates to a method for producing an antibody or fragment thereof that specifically binds to a region in an Aβ-peptide capable of being nitrated, in particular a 3NT10Aβ-antibody or fragment thereof, comprising the steps of a) affinity purification of a serum containing antibodies using a nitrated Aβ-peptide coupled to a chromatography column, or screening an sc-Fv phage display library using a nitrated Aβ-peptide, and b) optionally followed by a further purification step through exclusion of binding to a non-nitrated Aβ-peptide, such as a natural or recombinant Aβ-peptide lacking a tyrosine at position 10 thereof. The invention further relates to an anti-body or fragment thereof specifically binding to a region in an Aβ-peptide capable of being nitrated, in particular a 3NT10Aβ-antibody or fragment thereof, produced according to the method according to the present invention, wherein said antibody preferably is a monoclonal, polyclonal, human, humanized, and/or recombinant antibody or a functional fragment thereof.
The inhibitor of the aggregation of amyloid-β peptide according to the present invention is in a most preferred embodiment a substance specifically and exclusively interacting with the Aβ-peptide itself, preferably with a region in the Aβ-peptide capable of being nitrated, such as the tyrosine at position 10, thereby inhibiting Aβ-peptide nitration. Such an inhibitor is for example a specific antibody directed to the Aβ-peptide.
In a preferred third aspect of the present invention, the invention then relates to a pharmaceutical composition or formulation, and to a method for producing such a pharmaceutical composition, comprising an inhibitor as identified according to the present invention or an anti-body or fragment thereof according the present invention.
In a preferred fourth aspect of the present invention, the invention then relates to a recombinant non-human iNOS (−/−) mammal, in particular an APP iNOS (−/−) mouse, an APP (−/−) iNOS (−/−) mouse, or APP/PS-1 iNOS (−/−) mouse. Said animal can be used as a preferred advantageous “tool” in the context of the present invention.
In a still preferred fifth aspect of the present invention, the invention then relates to a diagnostic method for determining the status and/or progression of the aggregation of amyloid-β peptide (Aβ), comprising the steps of a) detecting the amount and/or fraction of nitrated amyloid-β peptide in a sample obtained from a patient to be diagnosed using an antibody or fragment thereof that specifically binds to a region in an Aβ-peptide capable of being nitrated, in particular a 3NT10Aβ-antibody or fragment thereof, b) comparing said amount and/or fraction of nitrated amyloid-β peptide as detected with a control sample, and, optionally, c) concluding on the status and/or progression of the aggregation of amyloid-β peptide (Aβ) based on said difference in the amount and/or fraction as detected between the sample and the control sample.
In a preferred sixth aspect of the present invention, the invention relates to a diagnostic kit, comprising an inhibitor as identified according to the present invention and/or an antibody or fragment thereof according to the present invention, optionally, together with additional auxiliary agents for performing a method according to an aspect of the present invention.
In a preferred seventh aspect of the present invention, the invention relates to an inhibitor as identified according to the present invention, an antibody or fragment thereof according to the present invention, an Aβ-peptide lacking a tyrosine at position 10 thereof, in particular a peptide selected from SEQ ID No. 2 or 3, or a pharmaceutical composition or formulation according to the present invention for use in the treatment of the aggregation of amyloid-β peptide (Aβ) and Alzheimers' disease.
Another embodiment of the above seventh aspect of the present invention relates to an Aβ-peptide lacking a tyrosine at position 10 thereof, in particular a peptide wherein the tyrosine at position 10 is substituted by an alanine or a phenylalanine, and respective pharmaceutical compositions and formulations thereof for use in the treatment of the aggregation of amyloid-β peptide (Aβ) and Alzheimers' disease.
In a preferred eighth aspect of the present invention, the invention then relates to a method for treating synaptic dysfunction and oxidative stress resulting in neuronal degeneration in a patient in need thereof, comprising administering a therapeutically effective amount of inhibitor as identified according to the present invention, an antibody or fragment thereof according to the present invention, an Aβ-peptide lacking a tyrosine at position 10 thereof, in particular a peptide selected from SEQ ID No. 2 or 3, or a pharmaceutical composition or formulation according to the present invention to said patient.
In the preferred ninth aspect of the present invention, the invention then finally relates to a method for improving cognitive functions in a patient suffering from neuronal degeneration, in particular from Alzheimers' disease, comprising administering a therapeutically effective amount of inhibitor as identified according to the present invention, an antibody or fragment thereof according to the present invention, an Aβ-peptide lacking a tyrosine at position 10 thereof, in particular a peptide selected from SEQ ID No. 2 or 3, or a pharmaceutical composition or formulation according to the present invention to said patient.
The present invention provides a method for identifying an inhibitor of the aggregation of amyloid-β peptide (Aβ), comprising the steps of a) contacting at least one Aβ-peptide and/or the nitrated forms thereof with at least one candidate inhibitor that potentially specifically binds to a region in said Aβ-peptide capable of being nitrated, and b) detecting said inhibitor specifically binding to said region in said Aβ-peptide through detecting a lack of or a reduced aggregation of said at least one Aβ-peptide.
The amino acid sequence of the human Aβ-peptide reads: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO. 1). This peptide comprises a site for nitration at the tyrosine at position 10 (bold and underlined). The fragments are generated from the (human) Aβ-precursor protein (APP) by subsequent cleavage of two aspartic proteases BACE1 and presenilin 1, resulting in the liberation of Aβ peptides of various lengths (Aβ 1-38/40/42). Thus, preferred is a method according to the present invention, wherein said Aβ-peptide is selected from 1-38, 1-40, and 1-42, and the nitrated forms thereof, and the term “Aβ-peptide” or “Aβ” is meant to include the Aβ-peptide selected from 1-38, 1-40, and 1-42, and the nitrated forms thereof, preferably Aβ-peptide 1-42. In one further preferred aspect of the method according to the present invention, said Aβ-peptide is a human Aβ-peptide.
Thus, further preferred is a method according to the present invention, wherein said region in said Aβ-peptide capable of being nitrated comprises a tyrosine at position 10 of said Aβ-peptide, and preferably is tyrosine at position 10 of said Aβ-peptide.
The amino acid sequence of the modified human Aβ-peptide according to the present invention reads: DAEFRHDSGAEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO. 2). This peptide comprises a mutation at the initial site for nitration at the tyrosine at position 10, which is replaced by an alanine (bold and underlined). Of course, other suitable amino acid replacements (i.e. other non-nitratable amino acids, such as phenylalanine) at this position are also encompassed by the term mutation according to the present invention. Another alternative is a chemically modified tyrosine, such as, for example, acetylation with Nacetylimidazole.
The amino acid sequence of the human Aβ-peptide reads: DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO. 3). This peptide comprises three differences compared to the human Aβ-peptide, one of which is present at the initial site for nitration at the tyrosine at position 10 (differences in bold).
Certain methods of screening are known in the art and are discussed, e.g., in: In vitro Methods in Pharmaceutical Research, Academic Press, 1997; and in U.S. Pat. No. 5,030,015. Preferred is a method for screening according to the present invention, wherein said potentially specific inhibitor is present in a compound library, a phage display library, in particular an sc-Fv phage display library, or in a library of antibodies. These libraries, their production and their screening in order to identify an inhibitor of the aggregation of amyloid-β peptide (Aβ) are known to the person of skill. Some libraries can be bought commercially and screened using machinery, such as robots.
The term “aggregation of amyloid-β peptide” shall mean the formation of aggregates of the amyloid-β peptide leading to plaques. Assays to determine the aggregation are described herein and well known from the respective literature, for example as cited herein.
Another aspect of the present invention then relates to the inhibitor screened according to the method according to the present invention. This inhibitor, according to the present invention, can be formulated into a pharmaceutical composition in a method for producing a pharmaceutical composition, comprising a method for identifying as above, and formulating said agent together with a pharmaceutically acceptable carrier, excipient, and/or stabilizer.
Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl-dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
Preferred is a method according to the present invention, wherein said inhibitor is selected from a compound selected from small chemical molecules, peptides, and antibodies and fragments thereof, in particular a natural or recombinant Aβ-peptide lacking a region capable of being nitrated, such as an Aβ-peptide lacking a tyrosine at position 10 thereof, in particular a peptide selected from SEQ ID No. 2 or 3, and an antibody or fragment thereof that specifically binds to a region in said Aβ-peptide capable of being nitrated, in particular a 3NT10Aβ-antibody or fragment thereof, which can be selected from a monoclonal, polyclonal, human, humanized, and/or recombinant antibody or a functional fragment thereof, optionally comprising a label.
In the context of the present invention, a “region” in said Aβ-peptide capable of being nitrated shall be a part of the amino acid chain where a nitration, either enzymatically (e.g. through iNOS, nNOS, and/or eNOS) or chemically, can take place. Examples for amino acids that undergo nitration are cysteine, methionine, tryptophan, and tyrosine. Nitration reactions Preferably, said region is found around the tyrosine at position 10 of the human Aβ-peptide, and includes N- and C-terminally located amino acids (for example, if the region constitutes an epitope for an antibody), such as 1, 2, 3, 4, 5 or 6 N- and C-terminally located amino acids around the tyrosine (i.e. FRHDSGYEVHHQ (SEQ ID NO. 4), SGYEV, or GYE in SEQ ID NO. 1). Furthermore, a region can also merely comprises the actual amino acid, that is, for example the tyrosine at position 10.
“Specifically binding” of the inhibitor shall mean that said substance exclusively or substantially exclusively binds and/or attaches to the region in said Aβ-peptide capable of being nitrated. Thus, incase of an antibody, said antibody shows no cross-reactivity or no substantial cross-reactivity with other antigens in the sample to be analyzed. One further example of specific binding is exclusive binding of, preferably, an antibody or functional fragment thereof, to the core of the Aβ-peptide plaque.
Preferred is a method according to the present invention, wherein said inhibitor inhibits the aggregation of said at least one Aβ-peptide through inhibiting nitration of said Aβ-peptide. That is, the formation of nitrated amino acids, such as 3′-nitrotyrosine is reduced or even completely inhibited. Another strategy is the inhibition of the aggregation of said at least one Aβ-peptide independently from the nitric oxide synthase (iNOS, nNOS, and/or eNOS) activity through blocking of the aggregation of the nitrated Aβ-peptide at the position of the nitration (preferably sterically). Yet another option would be the further chemical modification of the 3′-nitrotyrosine through the inhibitor, leading to a reduced or even completely inhibited aggregation.
Preferred is a method according to the present invention, wherein said method is performed in vivo in a recombinant non-human iNOS (−/−) mammal, in particular an APP iNOS (−/−) mouse, an APP (−/−) iNOS (−/−) mouse, or APP/PS-1 iNOS (−/−) mouse. The mouse is a convenient tool in order to further screen, identify and study prospective inhibitors of the aggregation of the at least one Aβ-peptide. In addition, modified Aβ-peptide (such as the natural mouse Aβ-peptide, and recombinantly modified human Aβ-peptides) and their effects independently of iNOS activity can be studied.
As mentioned above, a particularly preferred inhibitor according to the present invention is selected from antibodies and fragments thereof, in particular an antibody or fragment thereof that specifically binds to a region in said Aβ-peptide capable of being nitrated, in particular a 3NT10Aβ-antibody or fragment thereof, which can be selected from a monoclonal, polyclonal, human, humanized, and/or recombinant antibody or a functional fragment thereof, optionally comprising a label. Thus, the antibody or fragment thereof according to the present invention preferably has an immunoreactivity that is exclusively localized at the core of the Aβ-plaque.
Yet another important preferred aspect of the present invention then relates to a method for producing an antibody or fragment thereof that specifically binds to a region in an Aβ-peptide capable of being nitrated, in particular a 3NT10Aβ-antibody or fragment thereof, comprising the steps of a) affinity purification of a serum containing antibodies using a nitrated Aβ-peptide coupled to a chromatography column, or screening an sc-Fv phage display library using a nitrated Aβ-peptide, and b) optionally followed by a further purification step through exclusion of binding to at least one non-nitrated Aβ-peptide, such as a natural or recombinant Aβ-peptide lacking a tyrosine at position 10 thereof, such as the peptide according to SEQ ID No. 2 or 3. Respective details for these methods are known to the person of skill and as described herein, e.g. using a rabbit serum containing antibodies specific against a region in an Aβ-peptide capable of being nitrated, in particular a 3NT10Aβ-antibody or fragment thereof, for step a), or using natural or recombinant Aβ-peptides or parts thereof, such as a natural or recombinant Aβ-peptide lacking a tyrosine at position 10 thereof, such as the peptide according to SEQ ID No. 2 or 3 for step b).
Yet another important aspect of the present invention then relates to a pharmaceutical composition or formulation, produced according to a method according to the present invention as above containing the inhibitor as a diagnostic agent and/or therapeutic agent. Said pharmaceutical composition or formulation further contains a pharmaceutically acceptable carrier, excipient, and/or stabilizer. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl-dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
Yet another important preferred aspect of the present invention then relates to a recombinant non-human iNOS (−/−) mammal, in particular an APP iNOS (−/−) mouse, an APP (−/−) iNOS (−/−) mouse, or APP/PS-1 iNOS (−/−) mouse. The mouse is a convenient tool in order to further screen, identify and study prospective inhibitors of the aggregation of the at least one Aβ-peptide. In addition, modified Aβ-peptide (such as the natural mouse Aβ-peptide, and recombinantly modified human Aβ-peptides) and their effects independently of iNOS activity can be studied. In this context yet another important preferred aspect of the present invention relates to a recombinant non-human APP (−/−) mammal, in particular an APP (−/−) iNOS (−/−) mouse, or APP (−/−)/PS-1 iNOS (−/−) mouse. Preferably, said mouse further expresses a recombinant APP having a tyrosine at position 10 of the Aβ-peptide. Preferably, said recombinant APP having a tyrosine at position 10 is a modified mouse APP (i.e. a partially humanized APP). Since the modification of the mouse APP at position 10 appears to be sufficient in order to lead to an aggregation of the Aβ-peptide (since mice do not develop Aβ-peptide plaques, even when the murine Aβ-peptide is overexpressed), the recombinant non-human APP (−/−) mammal represents an extremely useful animal model to further study the aggregation of the Aβ-peptide and disease progression.
Yet another important preferred aspect of the present invention then relates to a diagnostic method for determining the status and/or progression of the aggregation of amyloid-β peptide (Aβ) in a mammal, particularly a human patient, comprising the steps of a) detecting the amount and/or fraction of nitrated amyloid-β peptide in a sample obtained from a patient to be diagnosed using an antibody or fragment thereof that specifically binds to a region in an Aβ-peptide capable of being nitrated, in particular a 3NT10Aβ-antibody or fragment thereof, b) comparing said amount and/or fraction of nitrated amyloid-β peptide as detected with a control sample, and, optionally, c) concluding on the status and/or progression of the aggregation of amyloid-β peptide (Aβ) based on said difference in the amount and/or fraction as detected between the sample and the control sample.
The sample obtained from said mammalian patient to be diagnosed can be derived from any suitable sample, such as whole blood, serum, plasma, urine, lymph fluid, brain liquor, tissue samples, such as brain tissue samples and/or biopsies, and prepared tissue samples, such as histological slides.
In a preferred diagnostic method for determining the status and/or progression of the aggregation of amyloid-β peptide (Aβ) in a mammal, particularly a human patient, according to the present invention, said method further comprises the step of concluding on the status and/or progression of Alzheimers' disease based on said status and/or progression of the aggregation of amyloid-β peptide (Aβ) as determined. Said status and/or progression of the aggregation can be measures in accordance with test known in the state of the art, and can comprise determinations of the amount of plaques, the proportion of plaques formed by amyloid-β peptide 1-40 and/or amyloid-β peptide 1-42, the localization of said plaques in the brain, and can further include the determination of cognitive functions of said mammal, as it is known to the person of skill.
Yet another important preferred aspect of the present invention then relates to a diagnostic kit, comprising an inhibitor as identified according to the present invention and/or an antibody or fragment thereof according to the present invention, optionally together with additional auxiliary agents for performing a method according to the present invention as above. The kit preferably contains the chemical substances, dyes, buffers, and the like that are required to perform the methods according to the present invention. The kit can also contain protein chips or microarrays for the analysis, as well as manuals and software and machinery in order to display and interpret the results of the diagnosis.
Yet another important preferred aspect of the present invention then relates to an inhibitor as identified according to according to the present invention, an antibody or fragment thereof according to according to the present invention, an Aβ-peptide lacking a tyrosine at position 10 thereof, in particular a peptide selected from SEQ ID No. 2 or 3, or a pharmaceutical composition or formulation according to according to the present invention for use in the treatment of the aggregation of amyloid-β peptide (Aβ) and preferably Alzheimers' disease.
Another important preferred aspect of the present invention then relates to a method for treating synaptic dysfunction and oxidative stress resulting in neuronal degeneration in a patient in need thereof, comprising administering a therapeutically effective amount of an inhibitor as identified according to the present invention, an antibody or fragment thereof according to the present invention, an Aβ-peptide lacking a tyrosine at position 10 thereof, in particular a peptide selected from SEQ ID No. 2 or 3, or a pharmaceutical composition or formulation according to the present invention to said patient.
“Treatment” as used herein refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder, in particular neuronal degeneration, aggregation of amyloid-β peptide (Aβ), and preferably Alzheimers' disease. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. The treatment can both include adjuvant treatments and first line treatments of treatment-naive patients, and can be combined with other anti neuronal degeneration strategies, such as chemotherapies. Preferably, said treatment in said patient is for the treatment of Alzheimers' disease.
Another important preferred aspect of the present invention then relates to a method for improving cognitive functions in a patient suffering from neuronal degeneration, in particular from Alzheimers' disease, comprising administering a therapeutically effective amount of an inhibitor as identified according to the present invention, an antibody or fragment thereof according to the present invention, an Aβ-peptide lacking a tyrosine at position 10 thereof, in particular a peptide selected from SEQ ID No. 2 or 3, or a pharmaceutical composition or formulation according to the present invention to said patient.
The inducible form of the nitric oxide synthase (iNOS), is transcriptionally upregulated in Alzheimer's disease. In the context of the present invention, the inventors determined the effect of iNOS deficiency in amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice. APP/PS1/iNOS (−/−) mice as well as APP/PS1 mice treated with the iNOS specific inhibitor L-NIL showed a significant reduction of working memory errors in the radial arm maze-test at 3 and 12 months of age as well as improvement of LTP at 3 months of age. Furthermore, APP/PS1/iNOS(−/−) mice revealed decreased amyloid β (Aβ) burden at 12 months, as detected by thioflavin S. Aβ 1-40 and 1-42 levels in brain extracts of these mice were reduced. This reduction could not be attributed to reduced microglial phagocytosis of Aβ. Instead, the inventors observed a decrease in the activity of insulin degrading enzyme (IDE) in APP/PS1 mice, which was rescued by iNOS gene deletion. IDE activity, in contrast to neprilysin, was also found to be specifically inhibited by nitric oxide in vitro. More importantly, the inventors observed that nitration of Aβ at tyrosine 10 strongly induces its aggregation. Raising a specific antibody against the Aβ(3NT-Y10) epitope the inventors were able to detect nitrated Aβ in plaques of APP/PS1 mice.
These results suggest that iNOS expression aggravates AD-like neuropathological changes, starting early with electrophysiological and behavioral phenotypes and ending with increased aggregation and decreased degradation of Aβ by IDE.
SEQ ID No. 1 shows the amino acid sequence of human Aβ1-42 peptide.
SEQ ID No. 2 shows the amino acid sequence of human Aβ1-42Y 10A peptide.
SEQ ID No. 3 shows the amino acid sequence of mouse Aβ1-42 peptide; and
SEQ ID NO. 4 shows the amino acid sequence of the region around T10 in human Aβ1-42 peptide.
SEQ ID NO. 5 shows the amino acid sequence of a mutated region around T10 in human Aβ1-42 peptide.